One of the goals of plant genetic engineering is to produce plants with agronomically, horticulturally or economically important characteristics or traits. Traits of particular interest include high yield, improved quality and high stability. Although the yield from a plant is influenced greatly by external environmental factors, it appears that the yield of the plant is determined, in part, by the intrinsic size of various organs/tissues (such as seeds, fruits, roots, leaves, tubers, stems, and bulbs) which are in turn determined by internal developmental factors. Enhancement of the yield of a plant may be achieved by genetically modifying the plant so that the intrinsic size of plant organs is increased.
Plants have unique developmental features that distinguish them from other eukaryotes. Plant cells do not undergo migration. It is thus believed that cell division and cell expansion are the predominant mechanisms by which the number and position of organ primordia are determined and also by which the intrinsic size and shape of each of plant organs are controlled. It is also believed that there are developmental regulators that control cell proliferation and growth and intrinsic size of plant organs. When interacting with external environmental factors, the developmental regulators determine the eventual size of plant organs. Therefore, identification/isolation of developmental regulators that control cell proliferation and growth and the intrinsic size of organs would be desirable. Such developmental regulators could be used in the genetic engineering to produce transgenic plants having increased intrinsic size of organs of interest and subsequently higher yield.
Gu et al.(Development 125:1509–1517 (1998)) recently reported that the Arabidopsis AGL8 gene, a MADS-box gene, might be involved in mediating cell differentiation in Arabidopsis plants during fruit and leaf development. Like AGAMOUS and other plant MADS-box genes, AGL8 encodes a polypeptide of about 260 amino acids including a highly conserved DNA-binding MADS domain of about 56 amino acids (Riechmann and Meyerowitz, Biol. Chem. 378:1079–1101 (1997)). They also reported that the ectopic expression of the AGL8 gene under control of a constitutive promoter in Arabidopsis plants could increase the size of seeds and fruits and delay senescence in the transgenic Arabidopsis plants (WO 99/00503).
The Arabidopsis APETALA2 (AP2) gene has recently been shown to be able to control seed mass in transgenic Arabidopsis and tobacco plants (WO 97/14659). The AP2 polypeptide contains two tandemly repeated 68-amino acid motifs designated as AP2 DNA binding domain (Jofuku, et al., Plant Cell 6:1211–1225 (1994), which are homologous to the DNA binding domain of ethylene response element binding polypeptides. Several studies suggested that the AP2 gene is a homeotic gene which controls three processes during flower development in Arabidopsis plants: (1) the establishment of flower meristem identity (Irish and Sussex, Plant cell 2:741–753 (1990); Bowman et al., Development 119:721–743 (1993)); (2) the specification of flower organ identity and regulation of floral organogenesis (Komaki et al., Development 104:195–203 (1988); Bowman et al., Plant Cell 1:37–42 (1989); Bowman et al., Development 112:1–20 (1991); Kunst et al., Plant Cell 1:1195–1208 (1989); Jofuku et al., Plant Cell 6:1211–1225 (1994)); and (3) the temporal and spatial regulation of flower homeotic gene activity (Drews et al., Cell 65:991–1002 (1991)). Genetic studies have shown that AP2 gene is also required for normal ovule and seed development (Jofuku et al., Plant Cell 6:1211–1225 (1994); Leon-Kloosterziel et al., Plant Cell 6:385–392 (1994); and Modrusan et al., Plant Cell 6:339–349 (1994)). Transgenic Arabidopsis plants, where the AP2 gene was expressed in the antisense orientation under the control of the cauliflower mosaic virus 35S constitutive promoter, produced seed with increased mass and total protein and fatty acid contents (WO 97/14659). Arabidopsis and tobacco transgenic plants, where the AP2 gene was overexpressed in the sense orientation under control of the cauliflower mosaic virus 35S constitutive promoter, produced seed with decreased mass and decreased total protein content (WO 97/14659).
It has been shown by two recent studies that the AINTEGUMENTA (ANT) gene of Arabidopsis might play a role in regulating cell growth and cell numbers during organogenesis (Mizukami and Fisher, Proc. Natl. Acad. Sci. USA 97:942–947 (2000); Krizek, Develop. Genet. 25:224–236 (1999)). The ANT gene belongs to the large AP2 gene family and encodes a transcription factor that may play a critical role in regulating ovule and female gametophyte development (Klucher et al., Plant Cell 8:137–153 (1996); Elliott et al., Plant Cell 8:155–168 (1996)). In one study (Mizukami and Fisher, Proc. Natl. Acad. Sci. USA 97:942–947 (2000)), it was reported that when the ANT gene was ectopically expressed in Arabidopsis plants under the control of a cauliflower mosaic virus 35S constitutive promoter, the leaves, stems, pedicels, sepals, petals, stamens, gynocia, ovules, and fruits of the transgenic plants were dramatically enlarged without altering their superficial morphology. Mass of leaves and flowers was increased as much as three times over those in control plants, due to the ectopic expression of the ANT gene. Ectopic expression of the ANT gene in tobacco plant also resulted in organs of increased size comparing to wild type. However, the transgenic plants containing a 35S/ANT expression construct were male sterile and most transgenic plants containing a 35S/ANT expression construct were also female sterile. Only T1 plants expressing relatively low levels of the ANT gene could generate seeds when pollinated by hand with wild-type pollen. In the other study, Krizek (Krizek, Develop. Genet. 25:224–236 (1999)) reported that ectopic expression of the ANT gene under the control of a cauliflower mosaic virus 35S constitutive promoter produced larger floral organs without altering the number and shape of these organs. The transgenic plants containing a 35S/ANT expression construct were male sterile and showed severe reduction in female fertility. Krizek did not observe the increased size of vegetative organs.
No DNAs encoding ANT-like polypeptides in other plants, especially corn, soybean, rice and cotton, have been isolated, sequenced or functionally characterized. Considering that the complex nature of organ size control in plants and that the genetic basis for plant interspecies diversity of phenotype might be minor changes in the structure or expression of orthologous regulatory genes (Doebley and Lukens, Plant Cell 10:1075–1082 (1998); Somerville and Somerville, Science 285:380–383 (1999)), there is a great deal of interest in identifying in plants the genes that, like ANT gene, may be used to control the intrinsic organ size of plants when ectopically expressed in plant cells and subsequently enhance the economic yield of plants.